Glutamate is the predominant excitatory transmitter in the central nervous system, and its cellular uptake is mediated by 5 excitatory amino acid transporters (EAAT1-5) that have distinct distribution patterns in mammalian brain. There are fundamental unanswered questions concerning the structure and function of glutamate transporters and their role in influencing the spatiotemporal profile of synaptically released glutamate. This application proposes to elucidate the molecular mechanisms underlying transporter gating and interaction with substrates, and to characterize the influence of the primary neuronal transporter (EAAT3) on synaptic transmission and synaptic plasticity in the hippocampus. The specific aims are: 1. To elucidate the mechanism and molecular pharmacology of glutamate transport by identifying and characterizing the structural determinants involved in controlling substrate selectivity and pore access. 2. To test the hypothesis that the neuronal transporter EAAT3 limits activation of extrasynaptic receptors at the Schaeffer collateral-CA1 synapse in hippocampus and influences postsynaptic responses, plasticity, and behavior. The results of these studies will be of significance in neurobiology and medicine because they will provide new tools and insights into the molecular mechanism of glutamate transport and its roles in the brain. Elucidating the transporters' structure, mechanism, and physiological functions is necessary for understanding how they function in normal and pathophysiological processes and for developing new therapeutic strategies for diseases like amyotrophic lateral sclerosis, epilepsy, and stroke.